Effects of Temperatures and Basal Media on Primary Culture of the Blastomeres Derived from the Embryos at Blastula Stage in Marine Medaka Oryzias Dancena

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including $28^{\circ}C$, $31^{\circ}C$, $34^{\circ}C$, and $37^{\circ}C$ and 2 basal media including Dulbecco's modified eagle's medium (DMEM) and Leibovitz's L-15 medium (L15). With the exception of a case cultured in L15 at $31^{\circ}C$, the rate of primary cell adherence reached 100% when the blastomeres were cultured over $31^{\circ}C$. The period for primary adherence was significantly shorter in the groups cultured in $34^{\circ}C$ and $37^{\circ}C$ than in the ones in $28^{\circ}C$ and $31^{\circ}C$. The proportion of subculture was significantly high in the group cultured in DMEM at $31^{\circ}C$ compared to the other groups. Collectively, we demonstrated that the culture in DMEM at $31^{\circ}C$ was effective to primary culture of the blastomeres derived from blastula embryos.